RESUMO
BACKGROUND: Uveal melanoma, the most common adult intraocular malignancy, metastasizes preferentially to the liver. Areas of cell death surrounding uveal melanoma metastases were observed in the livers of mice. We hypothesized that uveal melanoma cells might express Fas ligand (FasL), facilitating FasL-mediated apoptosis of Fas-expressing hepatocytes. PURPOSE: To determine whether Fas ligand (FasL)-expressing human uveal melanoma cells induce apoptosis of human hepatocytes in vitro and in vivo. METHODS: Human uveal melanoma cell lines were assayed for FasL expression by flow cytometry and immunohistology. A human hepatocyte cell line was assayed for Fas expression by flow cytometry. Apoptosis of hepatocytes was detected by annexin V staining in vitro, and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) in vivo. RESULTS: Human uveal melanoma cell lines expressed FasL, as determined by flow cytometry and immunohistology. Human hepatocytes were Fas-positive by flow cytometry. In vitro, annexin V staining revealed that human uveal melanoma cells induced apoptosis of human hepatocytes. TUNEL staining of liver metastases revealed apoptosis of murine hepatocytes in contact with metastatic human uveal melanoma cells. CONCLUSION: FasL-induced apoptosis of hepatocytes in contact with FasL-positive human uveal melanoma cells may contribute to hepatic failure during metastatic disease.
Assuntos
Apoptose , Neoplasias Hepáticas/secundário , Melanoma/secundário , Glicoproteínas de Membrana/fisiologia , Neoplasias Uveais/patologia , Animais , Anexina A5/metabolismo , Proteína Ligante Fas , Citometria de Fluxo , Hepatócitos/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Hepáticas/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Neoplasias Uveais/metabolismo , Receptor fas/metabolismoRESUMO
Human uveal melanoma arises in an immune privileged ocular environment in which both adaptive and innate immune effector mechanisms are suppressed. Uveal melanoma is the most common intraocular tumor in adults and is derived from tissues in the eye that produce macrophage migration-inhibitory factor (MIF), a cytokine that has recently been demonstrated to produce immediate inhibition of NK cell-mediated lytic activity. Although NK cell-mediated lysis of uveal melanomas is inhibited in the eye, melanoma cells that disseminate from the eye are at risk for surveillance by NK cells. Moreover, uveal melanoma cells demonstrate a propensity to metastasize to the liver, an organ with one of the highest levels of NK activity in the body. Therefore, we speculated that uveal melanomas produced MIF as a means of escaping NK cell-mediated lysis. Accordingly, seven primary uveal melanoma cell lines and two cell lines derived from uveal melanoma metastases were examined for their production of MIF. MIF was detected in melanoma culture supernatants by both ELISA and the classical bioassay of macrophage migration inhibition. Melanoma-derived MIF inhibited NK cell-mediated lysis of YAC-1 and uveal melanoma cells. Cell lines derived from uveal melanoma metastases produced approximately twice as much biologically active MIF as cultures from primary uveal melanomas. Inhibition of NK cell-mediated killing by uveal melanoma-derived MIF was specifically inhibited in a dose-dependent manner by anti-MIF Ab. The results suggest that human uveal melanoma cells maintain a microenvironment of immune privilege by secreting active MIF that protects against NK cell-mediated killing.
Assuntos
Citotoxicidade Imunológica/imunologia , Imunossupressores/metabolismo , Células Matadoras Naturais/imunologia , Fatores Inibidores da Migração de Macrófagos/biossíntese , Melanoma/imunologia , Melanoma/metabolismo , Neoplasias Uveais/imunologia , Neoplasias Uveais/metabolismo , Animais , Inibição de Migração Celular , Sistema Livre de Células/imunologia , Humanos , Soros Imunes/farmacologia , Imunossupressores/antagonistas & inibidores , Imunossupressores/farmacologia , Fatores Inibidores da Migração de Macrófagos/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais CultivadasRESUMO
PURPOSE: To determine whether aqueous humor promotes cell death in cells involved in inflammatory responses. METHODS: Multiple immune cell types, most characteristically involved in inflammatory responses, were incubated for 24, 48, and 72 hours in the presence or absence of 50% aqueous humor. Promotion of cell death was assayed by staining for an early indicator of apoptosis. The percentage of cells undergoing apoptosis was measured by flow cytometry. To identify partially the apoptosis inducing factor, aqueous humor was pretreated with proteinase K to degrade protein. In other experiments, aqueous humor was fractionated by centrifugation on filters capable of separating molecules above and below 10 kDa or 30 kDa kilodaltons in size. RESULTS: Rabbit aqueous humor promoted apoptosis in a wide variety of immune cells, including lymphokine-activated natural killer cells, resting T cells, an activated T-cell line, RAW 264.7 and J774A0.1 monocyte-macrophage cell lines, and neutrophils. As previously shown, aqueous humor did not promote apoptosis of murine corneal endothelial cells. Apoptosis was also not induced in human corneal endothelium, mouse corneal epithelium, or iris/ciliary body cell lines. Instead, aqueous humor partially protected these ocular tissues from starvation-induced cell death. Pretreatment with proteinase K inhibited the apoptosis-inducing activity. Moreover, the apoptosis-inducing activity segregated with the aqueous humor fraction containing molecules less than than 10 kDa in size. CONCLUSIONS: These data show that aqueous humor contains a factor or factors that promote death of cells that participate in inflammatory processes. By contrast, ocular tissues, such as the corneal endothelium and iris/ciliary body, are impervious to aqueous humor-induced cell death. The aqueous humor- borne factor(s) may contribute to the immune privilege of the anterior chamber by purging potential inflammatory cells.
Assuntos
Apoptose , Humor Aquoso/fisiologia , Células Matadoras Ativadas por Linfocina/fisiologia , Macrófagos/fisiologia , Neutrófilos/fisiologia , Linfócitos T/fisiologia , Animais , Apoptose/fisiologia , Linhagem Celular , Sobrevivência Celular , Citometria de Fluxo , Células Matadoras Ativadas por Linfocina/citologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Neutrófilos/citologia , Coelhos , Linfócitos T/citologiaRESUMO
PURPOSE: To determine if factors present in the aqueous humor (AH) protect the corneal endothelium from apoptosis. METHODS: Mouse and human corneal endothelial cells were cultured in vitro, and apoptosis was induced by nutrient deprivation. AH and supernatant from iris/ciliary body (I/CB) cell cultures were tested for their effect on corneal endothelial cell apoptosis. The effect of I/CB supernatant on Fas, Bax, and Bcl-2 gene transcription was evaluated by Northern blotting. I/CB supernatant was subjected to proteinase analysis to identify the apoptosis inhibitory factor(s). RESULTS: Rabbit AH and supernatant from mouse I/CB cell cultures inhibited the apoptosis of mouse and human immortalized corneal endothelial cell lines. The inhibition of apoptosis was associated with an upregulation of Bcl-2 gene transcription and Bcl-2 protein expression. Bax gene expression was not significantly affected by I/CB cell supernatant. Induction of apoptosis by stimulation of the Fas receptor was unaffected by I/CB cell supernatant. Protease analyses indicated that the apoptosis inhibitory factor was a protein. CONCLUSIONS: The inability of corneal endothelial cells to undergo mitosis renders the corneal endothelium vulnerable to loss of physiological function through cellular attrition. However, a protein(s) produced by I/CB cells and present in the AH, upregulates Bcl-2 gene transcription and protects the corneal endothelial cells from apoptosis.